Method and kit for isolating DNA probes that bind to activated transcription factors
View Patent ↗Methods, arrays and kits are provided for rapidly and efficiently identifying and quantifying multiple different activated transcription factors in a biological sample at the same time. In one aspect, a method is provided for isolating DNA probes which bind to activated transcription factors, including the step of mixing a library of double stranded DNA probes with a sample containing activated transcription factors. The transcription factor probes that have bound to the activated transcription factors are isolated from the mixture via an agarose gel separation. The bound probes can be identified, for example, by using an array of hybridization probes.
1. A method for isolating DNA probes which bind to activated transcription factors in a biological sample, the method comprising:
taking a library of double stranded DNA probes, wherein each DNA probes each comprises a recognition sequence varying within the library, and the variation on the DNA probes in the library is based on its binding to at least two known transcrintion factors selected from the group consisting of AP1, AP-2, ARE, Brn-3, C/EBP, CBF, CDP, c-Myb, CREB, E2F-1, EFR, ERE, Ets, Ets-1/PEA3, FAST-1, GAS/ISRE, GATA, GRE, HNF-4, IRF-1, MEF-1, MEF-2, Myc-Max, NF-1, NFATc, NF-E1, NF-E2, NFKB, Oct-1, p53, Pax-5, Pbx1, Pit 1, PPAR, PRE, RAR, RAR (DR-5), SIE, Smad SBE, Smad3/4, SP1, SRE, Stat1, Stat3, Stat4, Stat4, Stat5, Stat6, TFIID, TR, TR(DR-4), USF-1, VDR (DR-3), HSE, and MRE; contacting a biological sample with the library of double stranded DNA probes under conditions where DNA probe-transcription factor complexes are formed between the DNA probes and activated transcription factors present in the biological sample;
separating DNA probe—transcription factor complexes from non-complexed DNA probes in the library using an agarose gel separation;
excising a portion of the agarose gel comprising the separated DNA probe—transcription factor complexes; and
isolating the DNA probes from the excised portion of the agarose gel.
2. A method according to claim 1 wherein one strand of the double stranded DNA probes comprises a detectable marker.
3. A method according to claim 1 wherein one strand of the double stranded DNA probes comprises a detectable marker at a 5′ end of the strand.
4. A method according to claim 1 wherein one strand of the double stranded DNA probes comprises biotin at a 5′ end of the strand.
5. A method according to claim 1 wherein each of the probes in the library has a recognition sequence greater than 35 base pairs in length.
6. A method according to claim 1 wherein each of the probes in the library has a recognition sequence greater than 40 base pairs in length.
7. A method according to claim 1 wherein the recognition sequence on each of the probes in the library is between 20 and 40 base pairs in length.
8. A method according to claim 1 wherein the recognition sequence on each of the probes in the library is between 25 and 35 base pairs in length.
9. A method according to claim 1 wherein the library comprises at least 2 different DNA recognition sequences.
10. A method according to claim 1 wherein the library comprises at least 5 different DNA recognition sequences.
11. A method according to claim 1 wherein the library comprises DNA recognition sequences for at least 5 different types of cells.
12. A method according to claim 1 wherein the library comprises DNA recognition sequences for malignant, benign, and normal cell types.
13. A method according to claim 1 wherein the biological sample is a nuclear extract.
14. A method according to claim 1 , further comprising: identifying which of the DNA probes in the library are isolated.
15. The method according to claim 1 , wherein the recognition sequences comprised on the DNA probes are known to bind to at least two transcription factors selected from the group consisting of NF-E1, NFKB, Ets, Ap1, p53 and c-Myb.